Dissection of flavocytochrome b2 — a bifunctional enzyme — into a cytochrome core and a flavoprotein molecule

Proteolytic Cleavage of Hansenula anomala Flavocytochrome b2 into Its Two Functional Domains

“…This result is in complete agreement with the three-globules tertiary structure model that we have previously proposed for native flavocytochrome b,, these three globules being linked together by two protease-sensitive bridges [3].…”

“…The cytochrome and flavodehydrogenase domains of flavocytochrome 6, were finally isolated after mild trypsinolysis of H . unomuku flavocytochrome b, but the resulting flavodehydrogenase molecule was a 'nicked' form [3]. When S. aureus V8 protease I is used as a proteolytic agent (this study) the partition of the two functional domains can also be obtained: a cytochrome core (nv8) is formed along with a flavodehydrogenase (FDH",).…”

“…These two functional domains, flavodehydrogenase and cytochrome, which spontaneously dissociate from each other when their connecting bridge cd is cleaved by trypsin, were isolated as stable, pure, molecular entities, termed FDH, and nT respectively [2, 3, 91. Unfortunately, this polypeptide bridge cd is not the part of the native chain which is most sensitive towards trypsin: the aa' zone is cleaved about 20-times faster [lo], so that FDHT was always found altered by this undesirable inner cleavage, which was achieved with a loss of about 20 amino acid residues [3].From a functional point of view, the first cleavage in aa' results in a 96 %loss of activity [I I]; the isolated 'nicked' FDH, entity itself only retains about 1 % of the ferricyanide reductase activity of native flavocytochrome b2 [lo]. These results did not elucidate whether such a low activity was due to the aa' cleavdge itself or to the release of cytochrome b2 core resulting from the second cleavage at cd.…”

“…These two functional domains, flavodehydrogenase and cytochrome, which spontaneously dissociate from each other when their connecting bridge cd is cleaved by trypsin, were isolated as stable, pure, molecular entities, termed FDH, and nT respectively [2, 3, 91. Unfortunately, this polypeptide bridge cd is not the part of the native chain which is most sensitive towards trypsin: the aa' zone is cleaved about 20-times faster [lo], so that FDHT was always found altered by this undesirable inner cleavage, which was achieved with a loss of about 20 amino acid residues [3].…”

Study of the Hansenula anomala yeast flavocytochrome‐b2–cytochrome‐c complex 1. Characterization of fluorescent Zn(II)‐substituted cytochrome c

“…It is able to form reversible complexes with up to four molecules of yeast or mammalian cytochrome c per tetramer, at least at low ionic strength [7,81, and cytochrome c has always been considered as its physiological electron-acceptor. It has been established that in each protomer of tetrameric flavocytochrome b, the native polypeptide chain is folded into three 'globules' [9], one of which is a 'bJike' cytochrome [lo, 111 and the two others form together the functional flavodehydrogenase domain [9, itively charged belt of the former (Lys-I 3,27,72,73) and the cluster of negative charges at or near the heme group in the latter. This model, however, is still hypothetical, and the differences in amino acid sequence between cytochrome c and flavocytochrome b, from various yeast species may control the binding of yeast cytochrome c to flavocytochrome b,.…”